Genomics of Alkaliphiles Pedro H. Lebre & Don A. Cowan* * Corresponding author- Don A. Cowan: [email protected] Abstract Alkalinicity presents a challenge for life due to a “reversed” proton gradient that is unfavourable to many bioenergetic processes across the membranes of microorganisms. Despite this, many bacteria, archaea, and eukaryotes, collectively termed alkaliphiles, are adapted to life in alkaline ecosystems and are of great scientific and biotechnological due to their niche specialization and ability to produce highly stable enzymes. Advances in next-generation sequencing technologies have propelled not only the genomic characterization of many alkaliphilic microorganisms that have been isolated from nature alkaline sources, but also our understanding of the functional relationships between different taxa in microbial communities living in these ecosystems. In this review, we discuss the genetics and molecular biology of alkaliphiles from an ‘omics’ point of view, focusing on how metagenomics and transcriptomics have contributed to our understanding of these extremophiles. Introduction- Alkaliphiles in the Metagenomics era Alkaliphiles are a diverse group of micro-organisms that are defined as being able to grow in high pH environments (≥9). These organisms reside in a range of extreme environments in which high alkalinity has been established through geological processes, such as the accumulation of CO2 and subsequent production of carbonate/bicarbonate-rich solutions in soda lakes, as well as transient biological events such as ammonification and sulfate reduction in soils (Grant, 2009). In these extreme niches, microbial communities are not only well adapted to alkalinity, but they must also cope with a range of other environmental stresses, including high salinity, low or high temperatures, and oxygen deprivation (Yumoto, 2007). Consequently, alkaliphiles are of great scientific and biotechnological interest due to their highly selective niche specialization and their ability to produce proteins that are stable across a wide range of extreme conditions (Grant et al. 1990). Since the first report of an alkaliphile genome sequence in 2000 (Takami et al., 2000), the number of sequenced genomes of alkaliphilic microorganisms has increased exponentially due to advances in next-generation sequencing technologies (Koboldt et al. 2013). Currently, the JGI genome portal (Nordberg et al. 2014) lists the sequences of 288 genomes from isolated microorganisms that have been characterized to grow in alkaline conditions. These genomes are distributed across 10 different phyla (Figure 1A), and are highly variable in terms of GC-content (from 26% to 74%) and genome size (from 1.6Mb to 11Mb) (Figure 1B). This high genetic variability, together with the range of habitats from which alkaliphiles have been isolated, reflects the functional diversity of alkaliphilic microorganisms. As such, no obvious trends can be found between the alkaliphilic phenotype and particular genetic features. The phylum Firmicutes, which includes the historically relevant species Bacillus halodurans C-125 and B. pseudofirmus OF-4 (Takami et al. 2000; Janto et al. 2011), represents the second largest fraction of sequenced genomes (83 genomes), most of which have a GC content below 50 %. By comparison, the JGI lists 114 Proteobacteria alkaliphile genomes, the majority of which have a high GC content (≥ 50%). These include Halomonas sp. GFAJ-1, which is capable of thriving in arsenic-rich environments and has been associated with arsenate detoxification (Wu et al. 2018). To date, only two genomes for alkaliphilic Cyanobacteria have been listed in the JGI database, despite the fact that members of this phylum play a crucial role as primary photoautotrophic producers in many alkaline environments (Zavarzin et al. 1999). The two genomes belong to the desiccation-tolerant Chroococcidiopsis thermalis PCC 7203, and Arthrospira platensis C1, which is cultivated at large industrial scale as a food product for both humans and animals (Cheevadhanarak et al. 2012). All 16 publicly available alkaliphilic archaeal genomes belong to the Euryarchaeota phylum, and the majority of these organisms have been isolated from highly saline fresh water environments. Genome sizes for these archaea vary between 1.8 Mb to 4.9 Mb, and 12 have a very high CG content (≥ 60%). High genomic GC content is a common feature of halophiles, and has been associated to adaptation mechanisms against UV-induced thymidine dimer formation and the consequent accumulation of mutations (Paul et al. 2008). Ten other alkaliphilic cyanobacterial genomes that are not listed in the JGI database have also been sequenced, including five additional Arthrospira species (Klanchui et al. 2017). Compared to prokaryotic alkaliphiles, there is a dearth of knowledge on the genomics alkaliphilic eukaryotes. Crucially the recent drive for culture-independent techniques such as metagenomics has led to in-depth studies into the composition, functional capacities and ecological impact of communities living in alkaline environments. To date, 153 metagenomes have been obtained from alkaline environments, the majority of which were sourced from saline and alkaline water (51 metagenomes), and serpentinite rock and fluid (40 genomes). Metagenomics studies have also been crucial in detecting eukaryotic life in high pH environments. Members of the genera Frontania and Lacrymaria, both ciliates, were reported to be found in four distinct alkaline environments, while diatoms of the class Fragillariophyceae were found in both alkaline and acidic habitats (Amaral- Zettler, 2012). Figure 1A – Distribution of the JGI-listed alkaliphile genomes across different phyla. The number of genomes for each phylum is indicated in brackets next to the name for that phylum. Figure 1B- GC content and genome size distributions of the 288 alkaliphile genomes listed in the JGI database. GC content is illustrated with the scatter plot, and units are expression as fractions. Points in the plot are colour-coded to differentiate the different phyla. Genome size distribution is represented by the line plot, and units are expressed in nucleotide base-pairs (bp). The Genomic Features of Alkaliphilic Microorganisms The increasing number of genomes for alkaliphiles allows for comparative genomic studies that reveal the unique genetic features of these organisms. Historically, bacteria from the genus Bacillus have been the target of substantial research on the adaptation to alkaliphily, with the genomes of representative strains B. halodurans C-125 and B. pseudofirmus OF4 being widely studied and characterized (Takami, 2011). Both genomes share a large percentage of genes (1510), as well as 80% conserved synteny and comparable origins of replication (Janto et al. 2011). In turn, the genomes of B. halodurans C-125 and B. subtilis were found to share a high number of gene clusters involved in the house-keeping functions such as motility and chemotaxis, sporulation, protein secretion, main metabolic pathways, and DNA replication (Takami et al. 2000). One big differential factor between these genomes is the number and type of transposable elements. B. halodurans contains 112 transposable elements divided into 27 distinct groups compared to the 10 transposable elements in B. subtilis, and all of these share significant sequence similarity to transposases and recombinases from species such as Rhodobacter capsulatus and Lactococcus lactis. In addition, B. halodurans C-125 contains 10 unique extracytoplasmic function factors that might play a role in adaptation to alkaline environments (Takami et al. 2000). A distinct feature of B. pseudofirmus OF4 is the presence of two resident plasmids that contain gene clusters for metal acquisition and metal resistance, including P-type metal ATPases, copper chaperones, and cadmium resistance transporters (Janto et al. 2011). Genomic differences between B. pseudofirmus and B. halodurans further hint at the more alkaliphilic nature of the former. For instance, B. pseudofirmus contains 13 cation/solute antiporters compared to five in B. halodurans, which might contribute to increased capacity to maintain pH homeostasis in the cytoplasm (Janto et al. 2011). This enrichment in proton/cation transporters can also be seen in genomes from other alkaliphiles (Figure 2). Another alkaliphilic bacillus, Oceanobacillus iheyensis HTE831, first isolated from deep-sea sediments on the Iheya Ridge (Takami 2002), is a strict aerobe that grows optimally at pH 9.5. This organism contains a 3.6 Mb circular genome with 35.7% GC content, similar tRNA arrangement to B. subtilis and no prophages. This genome also contains 3496 CDSs. O. iheyensis and B. halodurans share 243 putative proteins. Five of these genes encode branched-chain amino acid transporters, which are thought to be important in alkaliphiles due to the conversion into negatively charged glutamic acid, which in turn leads to the acidification of the cytoplasm (Schadewaldt et al. 1995). This idea is further re- enforced by a recent transcriptomics study that shows the up-regulation of branched-chain amino acid transporters in Halomonas sp. Y2 under alkaline stress (Cheng et al. 2016). The obligately haloalkaliphile Natrialba magadii is an archaeon that requires a high salt concentration (3.5 M NaCl) and pH (9.5) for optimal growth. The genome consists of four replicons, the largest of which is 3.7Mb in size and has a high GC content (61.42%). Of the 4212 genes coded by the combined